Elsevier

Brain Research

Volume 1752, 1 February 2021, 147220
Brain Research

NLRP3 deficiency-induced hippocampal dysfunction and anxiety-like behavior in mice

https://doi.org/10.1016/j.brainres.2020.147220Get rights and content

Highlights

  • Deletion of NLRP3 inflammasome cause anxiety-like behavior in mice.

  • NLRP3 knockout mice exhibit abnormal behavior in the fear-conditioning test.

  • The adult neurogenesis in the dentate gyrus of NLRP3 knockout mice is impaired.

  • NLRP3 deletion leads to altered synaptic properties in hippocampal and amygdala neurons.

Abstract

Neuroinflammation has been classified as a trigger of behavioral alterations and cognitive impairments in many neurological conditions, including Alzheimer's disease, major depression, anxiety and others. Regardless of the cause of neuroinflammation, key molecules, which sense neuropathological conditions, are intracellular multiprotein signaling inflammasomes. Increasing evidence shows that the inflammatory response, mediated by activated nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasomes, is associated with the onset and progression of a wide range of diseases of the CNS. However, whether the NLRP3 inflammasome in the CNS is involved in the learning, development of anxiety and adult neurogenesis remains elusive. Therefore, the present study was designed to assess NLRP3 inflammasome contribution in anxiety and reveal its potential involvement in the experimental acquisition of fear responses and hippocampal neurogenesis. Behavioral, immunohistochemical and electrophysiological alterations were measured to evaluate role of neuroinflammation in the limbic system of mice.

In this study, we describe interrelated neurophysiological mechanisms, which culminate in absence of NLRP3 inflammasome in young 4 months mice. These include the following: anxious behavior and deterioration in learning and memory of fear conditioning; impairment of adult neurogenesis; reduction and altered morphology of astrocytes in the brain; hyperexcitability in basolateral amygdala (BLA); impaired activation in axons of pyramidal cells of CA1 hippocampal zone in NLRP3 KO mice particularly via the Schaffer collateral pathway; and impaired synaptic transduction in pyramidal cells mediated by an embarrassment of neurotransmitter release from presynaptic site in CA3 hippocampal zone.

The present study has demonstrated the novel findings that basal level of NLRP3 inflammasome in the brain of young mice is required for conditioning-induced plasticity in the ventral hippocampus and the basolateral amygdala. The deletion of NLRP3 impair synaptic transduction and caused anxiety-like behavior and labored fear learning, suggesting that low grade inflammation, mediated by NLRP3 expression, play a key role in memory consolidation.

Introduction

Behavioral and cognitive impairments are common in different neurological disturbances accompanied by neuroinflammation (Fellner et al., 2017, Wickens et al., 2018). These deteriorations often include memory impairments, fear, increased anxiety and depression-like behavior (Czerniawski and Guzowski, 2014, Dong et al., 2020, Fang et al., 2010, Patki et al., 2013, Yue et al., 2017). Although many studies regarding anxiety and fear have been published in recent decades, the exact pathophysiological mechanisms of anxiety and experimental acquisition of fear responses are not well delineated (Tovote et al., 2015). It should be noted, the experimental acquisition of fear responses serves as a competent model for studying memory and associative learning. Since memory and anxiety impairment is observed in many pathologies of the central nervous system, these processes have been studied to develop novel strategies to improve brain function, neuroscientists are studying the neural substrates and mechanisms that underlie fear and anxiety in animal models of normal and pathological brain function (Fadok et al., 2018).

Increasing evidence suggests an involvement of neuroinflammatory pathways in the pathophysiology of anxiety and cognitive decline (Leonard and Maes, 2012). Inflammation is a mandatory attribute of immune surveillance and protection, but chronic inflammation is a pathological feature of a wide range of chronic conditions such as neurodegenerative diseases, metabolic syndrome, type 2 diabetes mellitus, and others (Minihane et al., 2015). The inflammation is of decisive importance in maintaining the physiological homeostasis of the body and is initiated by the presentation of various pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Pathogen agents, cell-derived fragments and cellular dysfunction cause inflammatory responses. (Slowik et al., 2018). It is well known, that inflammatory responses are mandatory for eliminating pathological agents, clearing damaged cells and promoting tissue repair (Szretter et al., 2009); however, uncontrolled neuroinflammation may lead to further tissue injury and neuronal dysfunction (Capuron and Miller, 2011, Slowik et al., 2018). Thus, inflammation plays an important role in the body's homeostasis, protects it from disease, and aids recovery. On the other hand, long-term, uncontrolled chronic inflammation results in the release of pro-inflammatory cytokines and can expose the body to adverse conditions (Rönnbäck and Hansson, 2019).

Neuroinflammation has been classified as a trigger of behavioral alterations and cognitive impairments in many neurological conditions, including Alzheimer's disease, major depression and others (Capuron and Miller, 2011, Chen et al., 2019). The elevated expression of proinflammatory cytokines, increased microglial activation, especially in the limbic system, has been identified as a crucial contributor to neuroinflammation in neurodegeneration, chronic stress, and depressed suicide victims (Haroon et al., 2012, McGinnis et al., 2017).

Therefore, it has become evident that neuroinflammation represents a significant cause of neurological deficits. Regardless of the cause of neuroinflammation, key molecules, which sense neuropathological conditions, are intracellular multiprotein signaling inflammasomes (Slowik et al., 2018). It is known that inflammation is a central component of innate immunity. One of the canonical pathways of innate immune response is the activation of the NOD-like receptor (NLR) family, a pyrine domain containing 3 (NLRP3) inflammasome, which has been the subject of intense research (Venegas and Heneka, 2019). NLRP3 is a tripartite protein that consists of an amino-terminal pyrin domain (PYD), a central nucleotide-binding and oligomerization domain (NOD), and a C-terminal leucine-rich repeat (LRR) domain (Kelley et al., 2019). NLRP3 is abundantly expressed in the central nervous system (CNS) and may detect noxious agents in the cellular microenvironment (Yang et al., 2014). NLRP3 inflammasome activation is associated with the onset and progression of a wide range of diseases of the CNS, such as Alzheimer's disease (AD), Parkinson's disease (PD), anxiety (Dong et al., 2020, Patki et al., 2013, Venegas and Heneka, 2019, Zhang et al., 2019). When stimulated by PAMPs and DAMPs, NLR forms a protein complex with procaspase-1 and with or without assistance an adapter molecule such as an apoptosis-associated speck-caspase recruitment domain (ASC) (Dong et al., 2020, Venegas and Heneka, 2019). Activated NLRP3 inflammasome triggers the activation of caspase-1, which mediates the production of pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) and the initiation of a rapid form of cell death – pyroptosis (Fink et al., 2008). Therefore, the NLRP3 inflammasome plays a key role in the development of inflammatory responses in the CNS. Moreover, emerging studies have revealed the involvement of NLRP3 signaling in several neurological disorders (Johann et al., 2015). Neurodegenerative diseases are always accompanied by chronic neuroinflammation with the hyperproduction of IL-1β and IL-18 pro-inflammatory cytokines, which has destructive consequences for brain structure and function. The NLRP3 inflammasome has distinct importance in the development of inflammatory responses (Song et al., 2017).

Several lines of evidence indicate an important role for the NLRP3/caspase-1 axis in the pathogenesis of neurodegeneration, and suggest that NLRP3 inflammasome is activated in Alzheimer’s disease and contributes to pathology. It supports a fundamental role for NLRP3/caspase-1 mediated inflammation in behavioral and cognitive dysfunction in chronic pathology of the brain (Heneka et al., 2013). However, there are studies showing that neuroinflammation modulates synaptic plasticity in the hippocampus by influencing long-term potentiation. It was confirmed that cytokines influence synaptic plasticity and it is also supported by behavioral evidence that central cytokine signaling influences learning in several paradigms, including fear conditioning (Szczytkowski et al., 2013). Thus, acute administration of either TNF-α or IL-1β and chronic administration of interleukin-6 (IL-6) enhanced memory performance in a passive avoidance paradigm (Brennan et al., 2004). Furthermore, it is still not clear which effect drives cytokines, because some researchers report memory improvement (Depino et al., 2004), whereas others have reported memory impairment (Jones et al., 2015, Yirmiya et al., 2002).

In addition, there are a number of studies indicating that the cytokines are involved in both short- and long-term changes in synaptic plasticity (Lynch, 2015). However, whether the NLRP3 inflammasome in the CNS is involved in the learning, development of anxiety and adult neurogenesis remains elusive (Dong et al., 2020). Therefore, the present study was designed to assess NLRP3 inflammasome contribution in anxiety and reveal its potential involvement in the experimental acquisition of fear responses and hippocampal neurogenesis. Behavioral, immunohistochemical and electrophysiological alterations were measured to evaluate role of neuroinflammation in the limbic system of mice.

Section snippets

Deletion of NLRP3 augmented anxiety-like behavior in mice

When the mice were 4 months, they were first tested for locomotor activity in the open field arena. No significant difference was observed on the total distance travelled in the open field in both WT and NLRP3 KO groups, reflecting that general locomotor activity was similar in all mice (Fig. 1A). However, NLRP3 deletion increases anxiety-related behavior in the open field paradigm (Fig. 1B-D). Indeed, the time spent in the center of the arena was significantly reduced in NLRP3 group compared

Discussion

In this study, we describe interrelated neurophysiological mechanisms, which culminate in absence of NLRP3 inflammasome in young 4 months mice. These include the following: anxious behavior and deterioration in learning and memory of fear conditioning; impairment of adult neurogenesis; reduction and altered morphology of astrocytes in the brain; hyperexcitability in BLA; impaired activation in axons of pyramidal cells of CA1 hippocampal zone in NLRP3 KO mice particularly via the Schaffer

Experimental animals

Male wild-type 10-week-old (WT) and age-matched male NLRP3 gene knockout C57BL/6 mice (NLRP3 KO or NLRP3-/-) were all introduced from The Jackson Laboratory (USA). Mice were housed in a standardized animal room (lights on 7a.m.–7p.m.; room temperature 22 ± 2◦C), with food and water provided ad libitum. The groups are WT control group (n = 7) and NLRP3 KO group (n = 11). The experiment was carried out in accordance with the principles of humanity set forth in the European Community Directive

CRediT authorship contribution statement

Yulia K. Komleva: Conceptualization, Methodology, Visualization, Investigation, Data curation, Writing - original draft. Olga L. Lopatina: Investigation, Data curation. Iana V. Gorina: Investigation, Data curation. Anton N. Shuvaev: Investigation, Data curation. Anatoly Chernykh: Investigation. Ilia V. Potapenko: Investigation. Alla B. Salmina: Conceptualization, Validation, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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